CN109207577A - MARCO is screening the application in anti-blue otopathy pig - Google Patents
MARCO is screening the application in anti-blue otopathy pig Download PDFInfo
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Abstract
The invention discloses MARCO to screen the application in anti-blue otopathy pig.MARCO expression quantity and PRRSV infection are closely bound up in studies have shown that pig body of the present invention, inhibit MARCO expression after PRRSV infection PAMs, and MARCO interference promotes PRRSV duplication, and MARCO is overexpressed suppressing virus replication；When internal MARCO expression quantity is abnormal higher, pig is resistant to PRRSV, otherwise susceptible.Therefore, MARCO can be used as a kind of novel standard for determining PRRSV neurological susceptibility or resistance, solid foundation has been established to the screening and cultivation of PRRSV neurological susceptibility and PRRSV resistance pig to identify pig, screening and cultivation to PRRSV resistance pig have good clinical value, have great importance to the prevention and control of pig blue-ear disease, is also beneficial to cultivate excellent strain pig.
Description
Technical field
The invention belongs to biomedicine technical fields.Answering in anti-blue otopathy pig is being screened more particularly, to MARCO
With.
Background technique
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
PRRS), it is commonly called as blue otopathy, is by porcine reproductive and respiratory syndrome virus (Porcine reproductive
Andrespiratory syndrome virus, PRRSV) cause.Early in 1992, World Organization for Animal Health (Office
International Des Epizooties, OIE) disease is just classified as B class infectious disease.Currently, PRRS be in pig breeding industry most
One of important infectious disease is almost distributed in all pig-raising countries, causes huge economic loss to pig breeding industry in the whole world.
2006, porcine hyperthermia (Porcine high fever syndrome, PHFS) was broken out in China major part province and Vietnam,
Huge economic loss is caused to pig breeding industry, this disease is caused by the PRRSV to make a variation, is named as high pathological form pig breeding and is exhaled
Inhale syndrome virus (Highly pathogenic porcine reproductive and respiratory syndrome
Virus, HP-PRRSV).HP-PRRSV feature is high fever, highly pathogenic and high mortality.Currently, the disease is included in by the Ministry of Agriculture
Great animal epidemic plan of compulsory immunization.
The prevention and control of PRRSV are the problems in current China or even the world.PRRSV is difficult to prevention and control and is mainly manifested in following a few sides
Face: (1) thermophilic phagocytic and inhibitive ability of immunity disease, the pulmonary alveolar macrophage (Porcine of PRRSV main infection pig
Alveolar macrophages, PAMs), PAMs is immunocyte, PAMs is destroyed, so that body immune system is destroyed, thus
Cause immunosupress；(2) antigenic variability, PRRSV variation at present is very fast, and the use of attenuated vaccine is promote virus variation one
A reason, recently it has been reported that the new strain NADC30 of blue ear occurs in the U.S., and China is also separated to the new poison in the similar U.S.
Strain, is named as NADC30-like, and separately has document report to be separated to from pig farm and vaccine virus genome very high homology
PRRSV causes a disease strain, and virulence enhances, and it is anti-strong or recombinate and dissipate poison that analysis is likely to be vaccine virus；(3) vaccine does not intersect guarantor
Power is protected, PRRSV vaccine is almost without cross-protection currently on the market, not cross-protection between different strains；(4) antibody dependent
Enhancing, the infection of PRRSV can stimulate body to generate antibody, but the antibody of high-titer cannot not only neutralize virus, instead to virus
Proliferation have facilitation；(5) viral persistence infects, can be for a long time in pig vivo detection to viral blood after PRRSV infection
Disease, the duration is up to 5 months PRRSV in vivo；(6) mixed infection, clinically common blue ear is mixed with other diseases at present
Infection, the especially mixed infection of circovirus, haemophilus parasuis, swine plague etc. and PRRSV make in the prevention and control hardly possible of PRRSV
Add difficulty.
In addition, closely contacted since genetic background has with biological disease resistance, blue ear is screened based on gene means
The work of sick resistance pig has incomparable advantage for the prevention and control of the disease, it is preferred that emphasis is find out resistance or susceptible correlation
Gene, can not only study the infection mechanism of blue otopathy, the also treatment and prevention for blue otopathy provide new method.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defects and deficiency of existing pig blue-ear disease Prevention Technique, provide one kind
With pig blue-ear disease resistance related gene, the present invention is studies have shown that inhibit MARCO expression, MARCO after PRRSV infection PAMs cell
It is overexpressed and inhibits PRRSV duplication, MARCO interference promotes PRRSV duplication.The duplication breath of the expression quantity and PRRSV that show MARCO ceases
Correlation is expected to become a kind of method of anti-blue otopathy pig of novel screening, have to the cultivation of PRRSV resistance pig clinical well
Application value.
The object of the present invention is to provide MARCO to screen the application in anti-blue otopathy pig.
It is a further object of the present invention to provide a kind of judgement pigs to the method for blue otopathy resistance or neurological susceptibility.
Another object of the present invention is to provide MARCO in raising pig to the application in terms of the resistance of blue otopathy poison, or is making
It is standby to improve pig to the application in terms of the drug or preparation of blue ear virus resistance.
Another object of the present invention is to provide MARCO answering in terms of cultivating the pig variety resistant to blue otopathy poison
With.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The research of the invention finds that PRRSV infection PAMs cell different time points, different infection multiplicities inhibit MARCO in pig body
Expression.That is MARCO expression quantity and PRRSV infection is closely bound up, has close relationship to blue otopathy neurological susceptibility with pig, and pass through
PRRSV can be promoted to replicate after the expression of siRNA interference MARCO, MARCO inhibits PRRSV duplication after being overexpressed.I.e. in pig body
MARCO expression quantity is higher, then it is resistant to blue otopathy poison；Otherwise neurological susceptibility is higher.
Therefore, applying below should all be within protection scope of the present invention:
MARCO is screening the application in anti-blue otopathy pig.
The Gene ID:100516298 of the pig MARCO.
The indigo plant otopathy poison includes classical strains and highly pathogenic strain (HP-PRRSV).
MARCO is in raising pig to the application in terms of the resistance of blue otopathy poison.
MARCO improves pig to the application in terms of the drug or preparation of blue ear virus resistance in preparation.
Application of the MARCO in terms of screening and/or cultivating the pig variety resistant to blue otopathy poison.
MARCO expresses activator (as being overexpressed) in raising pig to the application in terms of blue ear virus resistance.
MARCO expresses activator and improves pig to the application in terms of the drug or preparation of blue ear virus resistance in preparation.
In addition, identifying pig to the method for blue otopathy neurological susceptibility, particular by detection pig body the present invention also provides a kind of
MARCO expression (expression quantity) is come to identify pig be resistance or neurological susceptibility to blue otopathy poison.
The standard of identification is: when MARCO expression quantity is high, pig is resistant to blue otopathy poison；Otherwise there is neurological susceptibility.
When specific operation, the height of MARCO expression quantity in art technology in pig body MARCO expression quantity general level
For foundation.Height described here be it is opposite, according to the higher or lower exception of MARCO expression quantity, therefore, it is determined that whether pig may be used
Can be resistant to PRRSV, to the neurological susceptibility degree of PRRSV.
More specifically, based on the present invention test as a result, the specific data standard of identification substantially are as follows: when MARCO expression quantity
Higher than 2.5 ± 0.5 × 10-2When (relative to internal reference HPRT1), pig is resistant to virus；Expression quantity is lower than 2.5 ± 0.5 × 10-2
It is susceptible when (relative to internal reference HPRT1).
The present invention does not limit the detection method of MARCO expression quantity strictly, can be existing gene in the art
Express quantity measuring method.
As a kind of preferable embodiment, the present invention provides a kind of primer sets for detecting pig MARCO expression quantity, packets
Upstream primer TREM2-F and downstream primer TREM2-R are included, sequence is respectively as shown in NO.1~2 SEQ ID.
The primer sets are determining pig to the application in terms of blue otopathy resistance or neurological susceptibility, or in screening anti-blue otopathy pig
Application, also all should be within protection scope of the present invention.
The present invention has selected different cultivars pig research MARCO screening the application in anti-blue otopathy pig.China is chosen respectively
Local varieties Tongcheng pig, plum mountain pig (not susceptible to blue otopathy) and external kind Landrace, Large White (susceptible to blue otopathy) are
Example carries out PRRSV and attacks poison, according to virus titer TCID in serum and lung, lymph node tissue after dissection50And PRRSV N protein table
Up to the determining Tongcheng pig of level, plum mountain pig and external kind Landrace, Large White to PRRSV infection power difference, expressed according to MARCO
Level studies it with 4 kinds of breeding pigs to PRRSV infection power relationship.The results show that of the present invention screen anti-blue ear using MARCO
The method of sick pig is reliable, accurate.
The invention has the following advantages:
The present invention studies for the first time has found that MARCO expression quantity is closely bound up with PRRSV infection in pig body, and MARCO can be used as one kind
The novel method for determining PRRSV resistance or neurological susceptibility；When MARCO expression quantity is abnormal higher, pig is resistant to PRRSV,
Otherwise it is susceptible.
It is of the invention to be found to be the anti-blue otopathy pig of screening and PRRSV breeding for disease resistance lays the foundation, to PRRSV resistance pig
Screening and cultivation have good clinical value, have great importance to the prevention and control of pig blue-ear disease, are also beneficial to excellent
The cultivation of strain pig.Meanwhile the present invention also provides a kind of new application for MARCO.
Detailed description of the invention
Fig. 1 is MARCO expression difference in different tissues (lung, spleen, liver, kidney).
Fig. 2 is PRRSV infection PAMs cell different time points MARCO mRNA expression.
Fig. 3 is PRRSV infection PAMs cell different time points MARCO protein expression level.
Fig. 4 is PRRSV infection PAMs cell difference infection multiplicity MARCO mRNA expression.
Fig. 5 is MARCO after siRNA is interfered, MARCO mRNA expression.
Fig. 6 is MARCO after siRNA is interfered, PRRSV N protein mRNA expression.
Fig. 7 is PRRSV N protein expression after MARCO is overexpressed.MYC is MARCO label.
Fig. 8 is PRRSV N protein mRNA expression after MARCO is overexpressed.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The statistical analysis of following embodiment of the present invention: all tests at least 3 times independent to repeat, as a result using average value and
Standard error indicates, is tested and analyzed using one-way analysis of variance and T.All statistical analysis are all made of aobvious using P < 0.05 as having
The test stone of statistical difference is write, analysis software is SPSS 16.0 and GraphPad Prism 5.
1 PAMs Cell isolation and culture of embodiment
1, the SPF grade of the specific pathogens such as no PRRSV, porcine circovirus 2 type (PCV-2), swine fever virus (CSFV) infection is detected
After the weanling pig binding of 6-8 week old, vena cava anterior bloodletting is lethal, ligatures tracheae, splits chest, takes out together with heart complete
Lungs sufficiently rinse lung surface with sterile PBS liquid, remove clot and dirt, extract heart after removing.Pour into PBS buffering
Liquid gently is patted to pinch and rubs lung surface 5-10 min, and BAL fluid is recycled.Step 2 is repeated to 3 times, until recycling altogether
About 1000 mL irrigating solutions.The bronchoalveolar lavage fluid that recycling obtains gently is blown and beaten back and forth with pipettor, separates cell mass, 4
DEG C, 1000 rpm are centrifuged 10 min of irrigating solution, discard supernatant.Cell is resuspended in 1640 culture medium of RPMI, and 4 DEG C, 1000 rpm are centrifuged
5 min, discard supernatant.It is gently blown and beaten with the RPMI-1640 nutrient solution of the FBS containing 20% and cell is resuspended, to prevent operation dirty
Dye, addition is dual anti-, isolated cell is uniformly assigned in culture plate, in 37 DEG C, 5% CO2It is cultivated in humidified incubator.
2, PAMs cellular morphology, activity are observed, Liquid nitrogen storage is spare.
The research of PRRSV duplication is adjusted outside 2 MARCO genosome of embodiment
1, with the RPMI 1640 culture medium culture PAMs cell for containing 10% fetal calf serum, malicious PRRSV is connect with MOI=0.1, in 2% tire
Continue to cultivate for 37 DEG C in the RPMI 1640 culture medium of cow's serum, culture different time points collect cell, respectively to MARCO gene
Do qRT-PCR and Western-blot detection.As a result as shown in Figure 2 and Figure 3, after PRRSV infection PAMs cell, MARCO expression quantity
It significantly reduces.
2, with the RPMI 1640 culture medium culture PAMs cell for containing 10% fetal calf serum, poison is connect with different infection multiplicity MOI
PRRSV continues to cultivate 24 h for 37 DEG C, collects cell, do to MARCO gene in the RPMI 1640 culture medium of 2% fetal calf serum
QRT-PCR detection.As a result as shown in figure 4, after PRRSV infection PAMs cell, MARCO expression quantity is significantly reduced.
3 MARCO of embodiment interference
1, designed MARCO siRNA and 1 pair of Negative Control (control) are sent to Thermo Fisher
The synthesis of Scientific company (is purchased from Thermo Fisher using Lipofectamine RNAiMAX transfection reagent
Scientific company).With the RPMI 1640 culture medium culture PAMs cell containing 10% fetal calf serum to 60- in 26 orifice plates
70% density respectively compares siRNA(3 to siRNA and 1) and Lipofectamine RNAiMAX Opti-MEM®
Reduced Serum Medium dilution is then added to and has changed newly in the mixing of 1:1 ratio and 5 min of incubated at room temperature after dilution
37 DEG C, 5%CO on the PAMs cell of fresh RPMI 1640 culture medium2It cultivates 6 h in incubator, abandons supernatant, PBS is washed 1 time, with containing
The RPMI 1640 culture medium of 10% fetal calf serum continues to cultivate 48 h, collects cell and is interfered by qRT-PCR experimental verification MARCO
Effect.After 48 h of another 6 orifice plates culture, PRRSV is added in the medium, is further cultured for 24 h, collects cell and passes through qRT-
PCR experiment replicates influence to PRRSV after verifying MARCO interference.
2, result is as shown in figure 5, MARCO is after siRNA is interfered, and relative to control group, siRNA is inhibited significantly
MARCO mRNA expression.
After MARCO interference, PRRSV connects poison, as a result as shown in fig. 6, relative to control group, after siRNA processing, and PRRSV N
Protein mRNA levels significantly increase.
In conclusion MARCO promotes PRRSV to replicate after siRNA is interfered.
4 MARCO of embodiment is overexpressed
1, pcDNA3.1-MARCO over-express vector is constructed, is cultivated in 26 orifice plates with the RPMI 1640 containing 10% fetal calf serum
Liquid culture PAMs cell (is purchased from Thermo Fisher using Lipofectamine 2000 to 60-70% density
Scientific company) pcDNA3.1-MARCO and pcDNA3.1 empty carrier is transferred to Marc-145 cell respectively, in 37 DEG C, 5%
CO26 h are cultivated in incubator, abandon supernatant, and PBS is washed 1 time, continues culture 48 with the RPMI 1640 culture medium containing 10% fetal calf serum
H collects cell detection and is overexpressed effect.After 48 h of another 6 orifice plates culture, PRRSV is added in the medium, is further cultured for 24
H, collecting to replicate PRRSV after cell detection MARCO is overexpressed influences.
2, for result as shown in fig. 7, after MARCO overexpression, MYC has expression, shows that MARCO is overexpressed successfully；Meanwhile significantly
Ground inhibits PRRSV N protein to express.
After MARCO is overexpressed, PRRSV connects poison, as a result as shown in figure 8, relative to control group, PRRSV N protein mRNA water
It is flat to significantly reduce.
In conclusion MARCO inhibits PRRSV duplication after being overexpressed.
5 MARCO of embodiment is screening the application in anti-blue otopathy pig
1, China's local varieties Tongcheng pig, plum mountain pig and external kind Landrace, Large White are chosen as experimental subjects, verifying is originally
The method that invention screens anti-blue otopathy pig using MARCO.
It is known that Tongcheng mountain Zhu Hemei pig is not susceptible to blue otopathy poison, i.e., it is resistant；And Landrace and Large White pair
Blue otopathy poison is more susceptible.
2, experimental method
Tongcheng pig, plum mountain pig, Landrace, Large White each 6 are selected, every 31 group, that is, is divided into 2 groups, wherein 1 group of carry out PRRSV
Poison is attacked, in addition 1 group (control) without any processing, 1 d and is attacked the 7th after poison, 14,21,28 d blood sampling, control before attacking poison respectively
The synchronous blood sampling of group, the 28th d are put to death, and acquire lung, lymph node tissue.
A part of serum is taken to carry out TCID50Measurement, remaining serum and lung, lymph node tissue extract RNA, reversion, to MARCO
And PRRSV N protein carries out qRT-PCR detection, and does pathological section to lung tissue.
According to TCID50, PRRSV N protein expression and pathologic be sliced and determine Tongcheng pig, plum mountain pig and foreign countries
Kind Landrace, Large White study it with 4 kinds of breeding pigs to PRRSV infection according to MARCO expression to PRRSV infection power
Power relationship.
3, it can determine whether according to result, Tongcheng mountain Zhu Hemei pig is not susceptible to blue otopathy poison, and Landrace and Large White are to indigo plant
Otopathy poison is more susceptible.
Meanwhile MARCO expression testing result is shown, MARCO expression is significantly high in the pig body of Tongcheng mountain Zhu Hemei
In Landrace and Large White.
In addition, show based on abundant experimental results, identification pig to the specific data standard of blue otopathy poison susceptibility substantially are as follows:
When MARCO expression quantity is higher than 2.5 ± 0.5 × 10-2When (relative to internal reference HPRT1), pig is resistant to virus；Expression quantity is lower than
2.5±0.5×10-2It is susceptible when (relative to internal reference HPRT1).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Zhongshan University
<120>MARCO is screening the application in anti-blue otopathy pig
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>upstream primer MARCO-F (forward primer MARCO-F)
<400> 1
gcagcgggta gacaacttc 19
<210> 2
<211> 20
<212> DNA
<213>downstream primer MARCO-R (reverse primer MARCO-R)
<400> 2
tgttgctcca tcttgtcccg 20
Claims (10)
- Application of the 1.MARCO in terms of screening anti-blue otopathy pig.
- 2. a kind of identify pig to the method for blue otopathy neurological susceptibility, which is characterized in that be by MARCO expression in detection pig body It is resistance or neurological susceptibility to blue otopathy poison to identify pig.
- 3. according to the method described in claim 2, it is characterized in that, the standard identified is: when MARCO expression quantity is high, pig is to indigo plant Otopathy poison is resistant；Otherwise there is neurological susceptibility.
- 4. a kind of primer sets for detecting pig MARCO expression quantity, which is characterized in that the primer sets include upstream primer TREM2-F With downstream primer TREM2-R, sequence is respectively as shown in NO.1~2 SEQ ID.
- 5. primer sets described in claim 4 are in judgement pig to the application in terms of blue otopathy resistance or neurological susceptibility.
- 6. primer sets described in claim 4 are screening the application in anti-blue otopathy pig.
- 7.MARCO is in raising pig to the application in terms of the resistance of blue otopathy poison.
- 8.MARCO improves pig to the application in terms of the drug or preparation of blue ear virus resistance in preparation.
- Application of the 9.MARCO in terms of cultivating the pig variety resistant to blue otopathy poison.
- 10.MARCO expresses activator in raising pig to the application in terms of blue ear virus resistance, or improves pig to blue otopathy in preparation The drug of malicious resistance or the application in terms of preparation.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810858762.3A CN109207577B (en) | 2018-07-31 | 2018-07-31 | Application of MARCO in screening of porcine reproductive and respiratory syndrome resistant pigs |
| PCT/CN2018/099287 WO2020024313A1 (en) | 2018-07-31 | 2018-08-08 | Application of marco in screening prrsv-resistant pigs |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810858762.3A CN109207577B (en) | 2018-07-31 | 2018-07-31 | Application of MARCO in screening of porcine reproductive and respiratory syndrome resistant pigs |
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| CN109207577B CN109207577B (en) | 2021-11-26 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804662A (en) * | 2019-09-30 | 2020-02-18 | 中山大学 | Method for screening anti-blue-ear disease pigs based on SIRT2 expression quantity |
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| CN117604167A (en) * | 2023-11-23 | 2024-02-27 | 扬州大学 | PMA-qPCR universal detection kit and triple identification kit for infectious PRRSV |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878104A (en) * | 2015-06-01 | 2015-09-02 | 北京泱深生物信息技术有限公司 | Bile duct cancer diagnosis and treatment molecular marker and application thereof |
| CN106244682A (en) * | 2016-07-29 | 2016-12-21 | 中山大学 | TREM 2 gene is differentiating that pig is to the application in reproductive and respiratory syndrome poison susceptibility |
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2018
- 2018-07-31 CN CN201810858762.3A patent/CN109207577B/en active Active
- 2018-08-08 WO PCT/CN2018/099287 patent/WO2020024313A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878104A (en) * | 2015-06-01 | 2015-09-02 | 北京泱深生物信息技术有限公司 | Bile duct cancer diagnosis and treatment molecular marker and application thereof |
| CN106244682A (en) * | 2016-07-29 | 2016-12-21 | 中山大学 | TREM 2 gene is differentiating that pig is to the application in reproductive and respiratory syndrome poison susceptibility |
Non-Patent Citations (1)
| Title |
|---|
| 黎倩倩: "猪A类清道夫受体SRA和MARCO的克隆、表达及其介导对病原菌吞噬功能的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804662A (en) * | 2019-09-30 | 2020-02-18 | 中山大学 | Method for screening anti-blue-ear disease pigs based on SIRT2 expression quantity |
| CN110804662B (en) * | 2019-09-30 | 2021-07-13 | 中山大学 | Method for screening anti-blue-ear disease pigs based on SIRT2 expression quantity |
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| WO2020024313A1 (en) | 2020-02-06 |
| CN109207577B (en) | 2021-11-26 |
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